Methods for reducing the incidence of necrotizing enterocolitis

ABSTRACT

Enteral formulas that contain long-chain polyunsaturated fatty acids (PUFAs), as arachidonic acid (AA) and docosahexaenoic acid (DHA) that are essentially free of cholesterol, are described. More particularly, the invention relates to methods for reducing the incidence of necrotizing enterocolitis by administering compositions which provide ω-6 and ω-3 long chain PUFAs, phospholipids and/or choline. Compositions from egg yolk lipids are presently preferred as they contain ω-6 and ω-3 long chain PUFAs and are predominantly in a phosphtidylcholine form. This is believed to provide a synergistic effect.

This application is a continuation-in-part of U.S. Ser. No. 08/804,700filed Feb. 21, 1997, abandoned, and of U.S. Ser. No. 08/825,314 filedMar. 28, 1997, now abandoned the entire disclosures of both of which areincorporated herein by reference.

Some aspects of this invention were developed in the Neonatal Nursery ofthe University of Tennessee Newborn Center under the direction of Dr.Susan E. Carlson with financial support from the Ross Products Divisionof Abbott Laboratories (Study AE78), NICHD grant RO1-HD31329, andNational Eye Institute grant RO1-EY08770. The U.S. Government may haverights to such aspects of this invention under NICHD grant RO1-HD31329and/or National Eye Institute grant RO1-EY08770.

TECHNICAL FIELD

The present invention relates generally to enteral formulas that containlong-chain polyunsaturated fatty acids (PUFAs) and to a process formaking such enteral compositions. More particularly, the presentinvention relates to enteral compositions which provide long chain PUFAsarachidonic acid (AA) and docosahexaenoic acid (DHA) essentially free ofcholesterol and may be derived from egg yolk lipids. Long chain PUFAsprovided from egg yolk are predominantly in a phospholipid form. Theprocess of making such a composition provides improved organoleptic andstability properties.

BACKGROUND OF THE INVENTION

Long chain PUFAs in enteral formulas or compositions have been thesubject of diverse literature. For example, U.S. Pat. No. 4,670,285("Clandinin") discloses a specific fat blend suitable for use in infantformulas. More specifically, the Clandinin fat blend contains at leastone C₂₀ or C₂₂ ω-6 fatty acid and a C₂₀ or C₂₂ ω-3 fatty acid. Thesefatty acids are disclosed as being at certain, defined amounts to avoidcausing harmful effects to an infant fed the fat blend. The C₂₀ or C₂₂ω-6 fatty acids are present in a total amount of about 0.13 to 5.6% byweight of all fatty acids in the product. The C₂₀ or C₂₂ ω-3 fatty acid,if present, are included in a total amount of about 0.013 to 3.33% byweight of all fatty acids in the product. Clandinin discloses the use ofegg lipids to supply the ω-6 and ω-3 fatty acids; however, the egg lipidused by Clandinin also contains high levels of cholesterol. Further,this reference teaches the use of 75 to 95 parts by wt. of egg yolklipid with the remainder of the oil being coconut oil or soybean oil.The nomenclature used by Clandinin for fatty acids will be utilizedherein.

WO 93/20717 discloses an infant formula which contains no more thansub-irritant amounts of free long chain (C16-C₂₂) fatty acids andtriglycerides. This application also discloses that providing loweralkyl esters, such as ethyl esters, of such fatty acids in infantformula essentially eliminates the tendency of the free fatty acid todamage the intestinal epithelium of the infant, but permits absorptionand processing of the fatty acid moiety.

U.S. Pat. No. 4,918,063 to Lichtenberger discloses compositionscontaining unique mixtures of phospholipids and neutral lipids for theprevention or treatment of ulcers and inflammatory bowel disease. Thispatent discloses mixtures of saturated or unsaturated phospholipids,together with saturated or unsaturated triglycerides and/or sterols, asproviding ulcer protective efficacy in experimental animal models. Thispatent also teaches the inclusion of polyvalent cations or antioxidantsto the lipid mixture to enhance activity.

International Publication No. WO 96/10922 to Kohn et al. discloses a fatmixture for infant formula characterized in that arachidonic acid anddocosahexaenoic acid are present in the fat mixture in the form ofphospholipids.

European Patent Application 0 376 628 B1 to Tomarelli discloses an allvegetable oil fat composition which utilizes randomized palm oil orrandomized palm olein oil as the sole palmitic acid oil source. It isalso disclosed that the all vegetable oil fat compositions areparticularly suited for use in infant formulas for pre-term (or lowbirth weight) infants. The pre-term fat compositions of the Tomarelliapplication include medium chain triglycerides (MCT's) with a randomizedpalmitic acid oil, lauric acid oil, an oleic acid oil and a linoleicacid oil.

Although the references discussed above have made importantcontributions, there remains a need for infant formulas that contain eggphospholipids as a source of long chain PUFAs in concentrationsappropriate for nutrition. A further need remains for methods ofpreparing enteral formulas containing egg phospholipids such that theformulas have acceptable organoleptic properties. Such compositions haveparticular application in infant formula for term and/or preterminfants, whose needs for long chain PUFAs are established for the properneural development and for development of visual acuity. In addition,there may be a protective effect on the gut.

Necrotizing enterocolitis (NEC) is a serious problem in infants havingbirth weights of less than about 1500 grams. Despite almost three (3)decades of study, the precise etiology and pathophysiology of NECremains unclear. NEC is a life-threatening disease characterized byischemic necrosis of the involved alimentary tract structures andpneumatosis intestinalis, which often results in the perforation of thebowel. A pre-term infant with NEC presents a clinical picture of thermalinstability, lethargy, gastric retention, vomiting, abdominaldistension, gross or occult blood in the stools and radiographicevidence of pneumatosis intestinalis, air in the portal veins orpneumoperitoneum. Apnea spells, shock and sclerema rapidly appear anddeath is common.

Numerous authors have made varied observations and posited factorsinfluencing this malady. (Nue, Pediatr. Clin. North. Am., April, 1996,43(2): 409-32). The following observations and factors are exemplary:

Flageole et al., Necrotizing Enterocolitis of the Newborn, Review forthe Clinician. Union-Med-Can. 1991 September-October; 120(5): 334-8,suggest the pathogenesis of NEC includes mesenteric ischemia,gastrointestinal immaturity, enteral feedings and even possiblyinfection;

Caplan et al., Role of Platelet Activating Factor and Tumor NecrosisFactor-Alpha in Neonatal Necrotizing Enterocolitis, Journal ofPediatrics, June, 1990, 960-964, report platelet activating factor andtumor necrosis factor-alpha are elevated in patients with NEC;

Kliegman et al., Clostridia as Pathogens in Neonatal NecrotizingEnterocolitis, The Journal of Pediatrics, August, 1979, 287-289, reportsthe isolation of Clostridia perfringens from children with neonatal NEC;

Ostertag et al., Early Enteral Feeding Does Not Affect the Incidence ofNecrotizing Enterocolitis, Pediatrics, Vol. 77, No. 3, March 1986,275-280, reports that dilute, early enteral calories do not adverselyaffect the incidence of NEC;

Bell et al., Neonatal Necrotizing Enterocolitis, Annals of Surgery, Vol.187, January 1978, No. 1, 1-7, suggests the use of combinationantimicrobial therapy for the treatment of infants with NEC;

Eyal et al., Necrotizing Enterocolitis in the Very Low Birth WeightInfant: Expressed Breast Milk Feeding Compared with Parenteral Feeding,Archives of Disease in Childhood, 1982, 57, 274-276 reports that theincidence of NEC in low birth weight infants was reduced by delaying theinitiation of enteral feeding.

Finer et al., Vitamin E and Necrotizing Enterocolitis, Pediatrics, Vol.73, No. 3, March 1984 suggests that administration of vitamin E toreduce the incidence of severe sequelae from retrolental fibroplasia maybe associated with an increased incidence of NEC.

Brown et al., Preventing Necrotizing Enterocolitis in Neonates, JAMA,Nov. 24, 1978, Vol. 240, No. 22, 2452-2454 reports that NEC can bevirtually eliminated by the use of a slowly progressive feeding regimen.

Kosloske, Pathogenesis and Prevention of Necrotizing Enterocolitis: AHypothesis Based on Personal Observation and a Review of the Literature,Pediatrics, Vol. 74, No. 6, December 1984, 1086-1092, hypothesizes thatNEC occurs by the coincidence of two of three pathological events: (1)intestinal ischemia; (2) colonization by pathogenic bacteria; and (3)excess protein substrate in the intestinal lumen. Kosloske, supra, alsoreports that NEC is rare among infants fed only breast milk. In humans,breast milk plays a role in passive immunization of the neonatalintestine, and contains factors that promote the growth ofBifidobacterium in the intestinal flora. It is also reported that thebeneficial contents of human milk may be adversely affected by freezing,pasteurization, or storage.

Thus, there is much debate about the etiology and treatment of NEC andthere remains a need for compositions and methods that are better ableto cure and/or reduce the incidence of this devastating and frequentlyfatal condition.

SUMMARY OF THE INVENTION

The present invention has many aspects. In a first aspect, the inventioncontemplates a method for reducing the incidence of necrotizingenterocolitis in an infant who is susceptible to necrotizingenterocolitis, said method comprising the administration of an effectiveamount of at least one long chain PUFA selected from the group of C₂₀ω-6 fatty acids, C₂₂ ω-6 fatty acids, C₂₀ ω-3 fatty acids and C₂₂ ω-3fatty acids. For example, the administration of arachidonic acid, an ω-6fatty acid, has been found to be effective. Administration may beenteral or parenteral. More preferably, a combination of ω-6 and ω-3fatty acids, for example arachidonic acid and docosahexaenoic acid, areused together. Enteral administration is at a level of at least 1.0 mgof ω-6 fatty acids per kilogram of infant weight per day. A morepreferred embodiment uses a combination of ω-6 and ω-3 fatty acids atweight ratios of about 2:1 to about 4:1 and administers at least 5.0 mglong chain ω-6 fatty acids per day.

The method may be performed by feeding a sufficient amount of an enteralcomposition containing arachidonic acid and docosahexaenoic acid todeliver to said infant about 1.0 to about 60 mg per kg per day ofarachidonic acid and about 0.25 to about 35 mg per kg per day ofdocosahexaenoic acid. More typical amounts are about 5.0 to about 40 mgarachidonic acid and about 1.5 to about 20 mg docosahexaenoic acid perkg per day. Preferably, the weight ratio of arachidonic acid todocosahexaenoic acid ranges from about 2 to about 4. Preferably, thelong-chain polyunsaturated fatty acids are in the form of phospholipids,especially phosphatidylcholine. Such phospholipids are present in highconcentrations in egg lecithin and egg phosphatides.

Thus, in a further aspect, the invention provides a method fordecreasing the incidence of necrotizing enterocolitis in an infant, saidmethod comprising feeding to said infant a sufficient quantity of anenteral nutritional composition containing protein, carbohydrate andphospholipids to provide at least 1.0 mg of ω-6 long-chainpolyunsaturated fatty acids per day. Preferably, the feeding furtherprovides at least 0.5 mg ω-3 long-chain polyunsaturated fatty acids perday and in the form of arachidonic acid and docosahexaenoic acid,respectively.

In another aspect, the invention provides a method for decreasing theoccurrence of necrotizing enterocolitis in a human infant, said methodcomprising administering to the infant phospholipids in an amounteffective to reduce the incidence of necrotizing enterocolitis.Typically said phospholipids are administered to provide between about60 and about 2400 μmoles, preferably between about 200 and about 1500μmoles, most preferably between about 400 and about 1000 μmoles ofphospholipids per kg day. The source of the phospholipids is notcrucial; phospholipids derived from egg lecithin are suitable for thisinvention. Phospholipids are also readily available from animalmembranes, including milk fat globule membrane. Other sources rich inphospholipids include soybean and other seed oils. When egg lecithinphospholipids are employed, the preferred amount to administer enterallyis enough to result in at least 1.0 mg of long chain ω-6 fatty acids perday. Preferably the egg phospholipid supplies arachidonic acid as asignificant portion of ω-6 fatty acids and preferably also suppliesdocosahexaenoic acid and/or other long chain ω-3 fatty acids in theratios mentioned above. This may have a synergistic effect. However, itis noted that the length and saturation of the fatty acids attached tothe glycerol backbone in this "phospholipid" aspect of the invention arenot crucial and fatty acids other than LCPUFAs may be employed here.

In yet another aspect, the invention provides a method for decreasingthe occurrence of necrotizing enterocolitis in a human infant, saidmethod comprising administering to the infant choline in an amounteffective to reduce the incidence of necrotizing enterocolitis.Typically said choline is administered to provide between about 60 andabout 1800 μmoles; more preferably between about 150 and about 1200μmoles of choline per kg per day. The source of the choline is notcrucial; phosphatidylcholine may be preferred and phosphatidylcholinederived from egg lecithin is suitable for this invention. Other sourcesrich in choline or phosphatidylcholine include soybean and other seedoils. When egg lecithin choline is employed, it is preferred toadminister in combination with ω-6 and/or ω-3 fatty acids to result inat least 1.0 mg of long chain ω-6 fatty acids per day. Preferably theegg lecithin supplies arachidonic acid as a significant portion of (ω-6fatty acids and preferably also supplies docosahexaenoic acid and/orother long chain ω-3 fatty acids in the ratios mentioned above. This mayhave a synergistic effect. However, as with the phospholipid aspect, thelength and saturation of the fatty acids attached to the glycerolbackbone of any phosphatidylcholine used in the invention are notcrucial and fatty acids other than LCPUFAs may be employed here.Alternatively, choline from non-phospholipid sources may be employed.

In addition, because of the beneficial effects seen in infantssusceptible to enterocolitis, it may well be the case that beneficialeffects are seen also in adults. Thus, a further aspect of the inventionis the use of any of the above compositions in treating or preventingulcerative colitis and related intestinal conditions in adults. Ofcourse dosing will be adjusted based on the increased weight of theadult patient and on other factors known in the art.

Other aspects of the invention, including enteral formulations andprocesses of making them, are described throughout the application. Forexample, in yet another aspect of the invention, a process for theproduction of an enteral formula comprising egg yolk phospholipidscomprises the steps of:

(a) providing dried egg phosphatide powder essentially free ofcholesterol;

(b) dispersing said phospholipid fraction in an aqueous phase to form aphospholipid dispersion; and

(c) combining said phospholipid dispersion with slurries of othercomponents of said enteral formula. Preferably according to the process,the dispersion in aqueous phase provides egg phosphatide at about 2 toabout 15 percent by weight; and preferably the egg phosphatide powder isadded to water at about 20 to 50° C. This aspect may be used to producean infant formula containing arachidonic acid and docosahexaenoic acidin the form of phospholipids, said enteral formula being produced by theprocess.

In yet another aspect, the invention provides a formula suitable forfeeding infants, comprising protein, carbohydrates and lipids, theimprovement characterized in a lipid blend comprising medium chaintriglycerides and egg phospholipid, wherein said egg phospholipid ispresent at a level from about 1 wt. % to about 40 wt. % of the lipidblend and wherein said egg phospholipid is essentially free ofcholesterol. Typically the egg phospholipid is present at a level offrom 5 to 30 wt. % of the lipid blend and the formula arachidonic acidin a concentration of from about 10 to about 31 mg per 100 kcals. Morepreferably, the formula also includes docosahexaenoic acid in aconcentration of from about 3 to about 16 mg per 100 kcals andarachidonic acid and docosahexaenoic acid are present in a ratio ofabout 4:1 to about 2:1.

DETAILED DESCRIPTION OF THE INVENTION

General Terminology

Fatty acids are hydrocarbon chains of various lengths, having acarboxylic acid at one end, thus making them somewhat polar andhydrophilic at this location, while being otherwise hydrophobic tovarying degrees depending on the length of the hydrocarbon chain. Fattyacids are categorized by the length of the hydrocarbon chain. Forexample, chains of fewer than about 6 carbons are considered "short";chains of about 6-18 carbons are "medium" and chains of 20 or morecarbons are considered "long". Fatty acids may also have one or moredouble bonds which are points of "unsaturation" in the hydrocarbonchain. As used herein, the term "long chain PUFA" means a fatty acid oftwenty carbon atoms or more having at least two carbon-carbon doublebonds (polyunsaturated). The number and position of double bonds infatty acids are designated by a convention of nomenclature. For example,arachidonic acid ("AA" or "ARA") has a chain length of 20 carbons and 4double bonds beginning at the sixth carbon from the methyl end. As aresult, it is referred to as "C₂₀ :4 ω-6". Similarly, docosahexaenoicacid ("DHA") has a chain length of 22 carbons with 6 double bondsbeginning with the third carbon from the methyl end and is designated"C₂₂ :6 ω-3". Less prevalent long chain PUFAs are also known and someare listed in Tables I and IV (below the solid line divider).

"Glycerides" are complex lipids having a glycerol backbone esterified tofatty acids. A "triglyceride" (i.e. "triacylglycerol") has threeesterified fatty acids, one to each hydroxyl site on the glycerolbackbone. Di- and mono-glycerides have, respectively, two and oneesterified fatty acid. A phosphoglyceride (i.e. "phospholipid" or"phosphatide"--all used interchangeably) differs from a triglyceride inhaving a maximum of two esterified fatty acids, while the third positionof the glycerol backbone is esterified to phosphoric acid, becoming a"phosphatidic acid". In nature, phosphatidic acid is usually associatedwith an alcohol which contributes a strongly polar head. Two suchalcohols commonly found in nature are choline and enthanolamine. A"lecithin" is a phosphatidic acid associated with the aminoalcohol,"choline", and is also known as "phosphatidylcholine". Lecithins vary inthe content of the fatty acid component and can be sourced from, forexample, eggs and soy. Cephalin (phosphatidylethanolamine),phosphatidylserine and phosphatidylinositol are other phosphoglycerides.

Phospholipids are commonly found in the membranes of all living systems.Traditional sources of phospholipids are egg yolk and soya bean oil.Phospholipids may also be obtained from mammalian brain, kidney, heartand lung; or from milk fat globule membranes. In addition, sources ofmicrobial origin (single cell oils) such as algal and fungal oils may beused, as well as genetically modified oil-bearing plants, particularlyfor the AA and DHA fatty acid components of phospholipids.

Hens' eggs are a relatively abundant source of lipids. Approximately 33%of the yolk of a hen's egg is lipid, of which about 67% is triglyceride,28% is phospholipid, and the remainder is mostly cholesterol(percentages are by weight). These figures are approximate and will varyto some degree, depending on the diet, breed and conditions of the hens.Of the phospholipid fraction, approximately 75% is phosphatidylcholine,and approximately another 20% is phosphatidylethanolamine. The cholinemoiety makes up about 15 to 30% of each phospholipid molecule dependingon the particular fatty acids attached. Thus, the choline content of eggphospholipid can vary from about 10% to about 25% by weight; or, on thebasis of total egg lipid, from about 3 to about 7% by weight.

Compositions

Compositions useful in the invention comprise ω-6 and/or ω-3 long chainPUFAs. The source of the long chain PUFA is not critical. Known sourcesof long chain PUFA include fish or marine oil, egg yolk lipid andphospholipids, single cell oils (e.g., algal oils and fungal oils), itbeing understood in the art that some sources are better than others forachieving higher amounts of specific long chain PUFAs. Other edible,semi-purified or purified sources of long chain PUFAs will be evident topersons skilled in the art. New sources of long chain PUFAs may bedeveloped through the genetic manipulation of vegetables and oil-bearingplants, and the use of such recombinant products is also contemplated inthe present invention.

The long chain PUFA may be provided in the composition in the form ofesters of free fatty acids; mono-, di- and tri-glycerides;phosphoglycerides, including lecithins; and/or mixtures thereof. It maybe preferable to provide long chain PUFAs in the form of phospholipids,especially phosphatidylcholine. A presently preferred source, at leastwhen processed such that the organoleptic properties and cholesterollevel are acceptable, appears to be egg yolk phospholipids, perhaps dueto the high phospholipid and/or phosphatidylcholine content associatedwith egg derived PUFAs.

The ω-6 and/or ω-3 fatty long chain PUFA may be administered in the formof an intravenous (i.e. parenteral) solution, as can choline andphosphatidylcholine. An intravenous solution will preferably containeffective amounts of the PUFA, the phospholipid and/or the choline in areasonable daily intake of parenteral solution. The exact concentration,therefore, is highly variable depending on the anticipated intake volumeand is significantly more concentrated in a bolus or small-volumeparenteral than in a hydrating or nutritional based parenteral product.Parenteral compositions will generally include pharmaceuticallyacceptable vehicles and excipients, such as buffers, preservatives, andthe like.

The ω-6 and/or ω-3 fatty long chain PUFA and the choline andphospholipid may alternatively be administered in the form of an enteralcomposition. Enteral compositions containing the long chain PUFA,choline or phospholipid may be in the form of a solution or an emulsionof active ingredient; or in a nutritional matrix comprising protein,carbohydrates, other fats, minerals and vitamins. Enteral compositionscontaining active components may provide either supplemental or completenutritional support. The concentration of the long chain PUFA in theenteral composition can range from almost 100% by weight (as in the caseof a bolus emulsion) to 0.5% by weight (as in the case of anutritionally complete formula) of the composition depending on the modeof administration and intended purpose. In complete nutritional formulasthe concentration may be even lower if enough of the formula isadministered to deliver effective amounts of the long chain PUFA.

A particularly preferred embodiment of this invention relates to animproved, nutritionally complete formula suitable for feeding toinfants, including pre-term infants. Such a preferred compositioncomprises protein, carbohydrates and lipids, wherein from about 6 toabout 40 wt. % of the total lipid is egg phospholipid which isessentially free of cholesterol. The term "essentially free" means thatthe cholesterol content of the egg phospholipid is less than 0.1 wt. %and preferably less than 0.05 wt. % of total lipid.

Those skilled in the art will readily understand what is meant by aninfant formula. When diluted or reconstituted, if initially inconcentrate or powder form, to the ready to feed state, a typical infantformula contains about 10-35 gms of protein per liter of formula; 20-50gms of lipid per liter of formula; 60-110 gms of carbohydrates per literof formula and other various components such as vitamins, minerals,fibers, emulsifiers and the like. For purposes of understanding thecomponents of an infant formula and methods for its production, thefollowing U.S. patents are herein incorporated by reference: 1) U.S.Pat. No. 5,492,899 to Masor et al.; 2) U.S. Pat. No. 5,021,245 toBorschel et al.; 3) U.S. Pat. No. 5,234,702 to Katz et al.; and 4) U.S.Pat. No. 5,602,109 to Masor et al.; and 5) U.S. Pat. No. 4,670,268 toMahmoud. More specifically, this embodiment of the invention comprehendsan infant formula containing about 40-50 gms of lipid per liter offormula wherein the lipid comprises a blend of medium chaintriglycerides and egg phospholipid that is essentially free ofcholesterol. Typically, the lipid blend comprises from about 1-40 wt. %,more preferably about 5 to about 30 wt. %, of the egg phospholipid. Thisembodiment is specifically designed to provide long chain PUFAs selectedfrom ω-3 fatty acids and ω-6 fatty acids, phospholipids, and/or cholinein amounts beneficial to infants.

Process of Making

Since hens' egg yolks include both triglycerides and phosphatides, itmay be preferable to process the egg yolks using organic solvents in amanner that separates the phosphatides from triglycerides, sterols (e.g.cholesterol) and other components. Various literature methods aresuitable for this separation, at least in laboratory scale.Alternatively, such egg phosphatides essentially free of cholesterol arecommercially available in dried powder form from Pfanstiehl, Inc.(Waukegan, Ill.) as Catalog No. P-123.

The egg phosphatide is then incorporated into the enteral composition ofthe present invention. Because of the lipid content, incorporation ofthe egg phosphatides into an enteral formula was expected to be facilein an oil phase. However, it was surprisingly discovered that theselipid-lipid dispersions were unacceptable and that preparation of anaqueous dispersion of the egg phosphatide resulted in improved product.Aqueous dispersions of about 2-15 wt %, preferably about 3 to about 8 wt%, should be made in cool to ambient water (about 20-25° C.) to providethe best results. Warmer water produced less acceptable organolepticproperties.

Separately, the carbohydrate, protein and lipid slurries that comprisethe macronutrient source are prepared as is known in the art, and theseslurries are mixed at about 130 to 140° C. Just prior to homogenization,the phosphatide dispersion is mixed with the remainder of the formula.

In a particularly preferred variation, prior to the addition of thephosphatide dispersion to the final product mix (just beforehomogenization) the phosphatide dispersion is de-aerated under amoderate vacuum. De-aeration may be effected by any mechanism but anatomizing de-aerator at about 15 inches Hg provided satisfactoryresults. This additional step has been shown to improve the organolepticand olfactory properties of the final product, even more so thanactivated carbon filtration or a combination of the two (see ExampleIII).

For making parenteral compositions useful in this invention,conventional sterile parenteral production technology may be used. Itmay be preferable in this case to avoid the egg phosphatides and employinstead the triglyceride oils or fatty acid esters, such as may be foundin recombinant or single cell oil sources.

Utility

Compositions of the present invention are useful in the nutritionalsupport of infants and/or adults. The addition of long chain PUFAs,especially ω-6 and ω-3 fatty acids and most especially AA and DHA havegenerally been considered to be beneficial to neural development andvisual acuity of the infant, although conflicting reports have also beenfound in the literature.

Compositions useful in the present invention include those containingany or all of the following:

(a) long chain PUFAs selected from ω-6 and ω-3 fatty acids, typically inphospholipid or phosphatidylcholine form;

(b) polar phospholipids, regardless of the nature or length of theattached fatty acids;

(c) choline, preferably phosphatidylcholine.

For example, the egg phospholipid fortified formula described in detailin the examples provided higher levels of each of the specifiedcomponents and surprisingly was found to reduce substantially theincidence of NEC in infant populations that are susceptible to NEC. In amore specific embodiment, the method of reducing the incidence of NEC isaccomplished through the administration of arachidonic acid (AA, 20:4ω-6) or, more preferably, AA in combination with docosahexaenoic acid(DHA, 22:6 ω-3).

More broadly, this aspect of the invention contemplates a method forreducing the incidence of necrotizing enterocolitis in an infant whichis susceptible to necrotizing enterocolitis, said method comprising theadministration of an effective amount of at least one long chain PUFAselected from the group of C₂₀ ω-6 fatty acids, C₂₂ ω-6 fatty acids, C₂₀ω-3 fatty acids and C₂₂ ω-3 fatty acids. The administration is at alevel of at least 1.0 mg of ω-3 fatty acids per kilogram of infantweight per day. A more preferred embodiment uses a combination of ω-6and ω-3 fatty acids at weight ratios of about 2:1 to about 4:1.

There is further disclosed a method for decreasing the occurrence ofnecrotizing enterocolitis in a human infant, said method comprisingadministering to the infant egg phospholipids in an amount to result inat least 1.0 mg of long chain ω-6 fatty acids per day. Preferably theegg phospholipids supplies AA as a significant portion of the ω-6 fattyacids and preferably also supplies DHA and/or other long chain ω-3 fattyacids in the ratios mentioned above.

An additional aspect of this invention relates to the enteraladministration to humans of phospholipids, especially phospholipidscontaining AA and/or DHA which readily increase the blood serum levelsof fatty acids AA and DHA in humans relative to compositions havingtriglycerides of AA and DHA.

A more appropriate measure of administration of compositions accordingto the invention is as daily intake in mg per kg infant body weight. Thefollowing Table A gives guidelines for minimum, preferred and idealtarget ranges for each of the compositions useful in the invention.

                                      TABLE A                                     __________________________________________________________________________    Daily Intake Guidelines                                                       (based on per kg infant weight)                                               Component  minimum                                                                            preferred                                                                             more preferred                                                                       ideal target                                   __________________________________________________________________________    AA, mg     1     2 to 60                                                                               5 to 40                                                                             10 to 30                                       DHA, mg    0.25 0.5 to 35                                                                             1.5 to 20                                                                             3 to 15                                       AA/DHA ratio                                                                             0.25 0.5 to 10                                                                             1 to 8 2 to 4                                         phospholipid, μmoles                                                                  50    60 to 2400                                                                            200 to 1500                                                                          400 to 1000                                   choline, μmoles                                                                       50    60 to 1800                                                                            150 to 1200                                                                         300 to 900                                     __________________________________________________________________________

There is a wide variance in the ranges largely due to the fact that notall infants who are likely to benefit from this invention will consumeequal volumes of formula. Those who consume less, received less of eachingredient. The ideal target ranges assume approximately 100 kcals willbe consumed. Also, there is controversy about the methods for estimatingthe choline content.

It can be seen from Table A that most preferably there is about 2 to 4times more AA than DHA. It is also observed that the minimum levels ofphospholipid and choline are identical. This is attainable by providingall the phospholipid as phosphatidylcholine. As other nitrogenousalcohols replace the choline, (eg. ethanolamine, serine or inositol) therelative amount of choline to total phospholipid decreases.

The AA and/or DHA can be administered individually, as separatecomponents, or together, or in combination with other ingredients suchas protein, lipid, carbohydrate, vitamins and minerals. Nutritionalsupport for low birth weight infants is either parenteral (intravenousfeeding) or enteral. Thus, the appropriate levels of long chain PUFA canbe incorporated into the parenteral nutrition solution or added to aconventional low birth weight enteral formula. Most preferably, themethod of the present invention is accomplished through the enteraladministration of an infant formula designed for low-birth weightinfants containing AA and DHA. Such an infant formula further comprisesappropriate levels of carbohydrate and protein and an appropriatecombination of minerals and vitamins. An exemplary infant formula foruse in the methods of the present invention is a modified SimilacSpecial Care® (Ross Products Division of Abbott Laboratories, Columbus,Ohio), which is discussed in more detail in Example II.

An additional aspect of this invention is a method for increasing theblood serum levels of arachidonic acid and docosahexaenoic acid in humanblood serum, said method comprising the step of administering to saidhuman an enteral formula containing AA and DHA in the form ofphospholipids.

Recent studies by the present Applicants have indicated that theadministration of long chain PUFA, to infants susceptible to NEC willreduce the incidence of NEC and may also reduce the level or severity ofNEC. The Applicants have also discovered that the administration ofphospholipids from animal or vegetable sources is also effective inreducing the incidence of NEC in infant populations that are susceptibleto NEC.

EXAMPLE I

Egg yolk phosphatide was obtained from Pfanstiehl, Inc. (Waukegan,Ill.--Catalog No. P-123) and was used in the following Examples. Thefatty acid and cholesterol profile of this egg phosphatide is set forthin Table I. The sum of all ω-3 and of all ω-6 "long chain" PUFAs is alsogiven.

                  TABLE I                                                         ______________________________________                                        Fatty Acid Profile and Cholesterol Content of Egg Yolk Lecithin               Fatty Acid       gm/100 gm of sample                                          ______________________________________                                        C14:0                       0.08                                              C16:0                      18.83                                              C16:1 ω-7             0.82                                              C16:4                       0.21                                              C18:0                       6.72                                              C18:1 ω-9            17.36                                              C18:2 ω-6            9.8                                                C20:1 ω-9             0.11                                              C20:2 ω-6                    0.24                                       C20:3 ω-6                    0.3                                        C20:4 ω-6 - arachidonic      4.93                                       C22:0                       0.07                                              C22:4 ω-6                    0.3                                        C22:5 ω-6                    1.45                                       C22:5 ω-3  0.09                                                         C22:6 ω-3 - docosahexaenoic                                                              1.24                                                         cholesterol                <0.05                                              Total LCPUFA ω-6             7.22                                       Total LCPUFA ω-3                                                                         1.33                                                         ______________________________________                                    

Those skilled in the art will appreciate that the specific levels of thevarious fatty acids contained in egg yolk lipid will vary depending onthe breed, diet and age of the hen. In addition, the extractionprocedure used by Pfanstiehl to prepare the phosphatide used in theExamples results in a material that contains extremely low levels ofcholesterol while possessing a fatty acid profile that is highly usefulin the nutritional arts.

EXAMPLE II

In this example, "Experimental" and "Control" infant formulas wereprepared, respectively, with and without the egg phosphatide of ExampleI. The Control composition was Similac Special Care®(Ross ProductsDivision of Abbott Laboratories, Columbus, Ohio) and was prepared usingthe following list of ingredients, which results in the formula havingthe composition set forth in Tables II-IV, below: Water (Kosher), nonfatmilk, hydrolyzed cornstarch, lactose, fractionated coconut oil(medium-chain triglycerides), whey protein concentrate, soy oil, coconutoil, calcium phosphate tribasic, potassium citrate, sodium citrate,magnesium chloride, ascorbic acid , mono- and diglycerides, soylecithin, calcium carbonate, carrageenan, choline chloride, ferroussulfate, m-inositol, taurine, niacinamide, L-carnitine, alpha-tocopherolacetate, zinc sulfate, calcium pantothenate, potassium chloride, cupricsulfate, riboflavin and vitamin A palmitate, thiamin chloridehydrochloride, pyridoxine hydrochloride, biotin, folic acid, manganesesulfate, phylloquinone, vitamin D₃, sodium selenite and cyanocobalamin.

Generally, protein, carbohydrate, lipid, vitamin and mineral slurriesare separately prepared and then these are mixed prior to homogenizationas is generally taught in the previously incorporated US patentsrelating to the manufacture of infant formula.

In the experimental formula, the egg phosphatide of Example I wasincorporated into the formula during manufacture. First, the eggphosphatide was dispersed in water at 25° C. to make an 8% dispersion.Just prior to homogenization the phosphatide dispersion was combinedwith the protein, carbohydrate, vitamin, mineral and other lipidslurries to result in an "Experimental" formula having the compositionshown in Tables II-IV, below. The amounts of each component are givenboth on a "per Liter" basis and on a "per kcal" basis since it is wellknown in the art to prepare infant formulas having higher or lowercaloric densities than the standard 20 kcal per fluid ounce.

                  TABLE II                                                        ______________________________________                                        COMPONENTS of CONTROL AND EXPERIMENTAL FORMULAS                                             Preferred Ranges                                                Nutrient        Units Per Liter*                                                                          Units per 100 kcal                                ______________________________________                                        Protein, g      21.9-23.4   2.61-2.88                                         Fat, g          40.0-46.0   5.24-5.67                                         (as described in Table III below)                                             Carbohydrate, g 84.0-88.0   10.00-10.84                                       [Ash, g]        6.7-8.0     0.80-0.99                                         Total Solids, g 158.6-165.0 18.88-20.32                                       Linoleic Acid, g                                                                               5.6-12.2   0.67-1.50                                         Calcium, mg     1300-1700   154.76-209.36                                     Phosphorus, mg  720-970      85.71-119.46                                     Magnesium, mg   100-170     11.90-20.94                                       Sodium, mg      349-389     41.55-47.91                                       Potassium, mg   1000-1420   119.05-174.88                                     Chloride, mg    650-770     77.38-94.83                                       Iron, mg        3.0-5.5     0.36-0.68                                         Zinc, mg        12.0-14.6   1.43-1.80                                         Copper, mg      2.0-3.0     0.24-0.37                                         Manganese, mcg  100-500     11.90-61.58                                       Iodine, mg      0.05-0.30   0.01-0.04                                         Selenium, mcg   12-29       1.43-3.57                                         Vitamin A, IU   6000-8000   714.29-985.22                                     Vitamin D, IU   1200-1580   142.86-194.58                                     Vitamin E, IU   35.0-45     4.17-5.54                                         Vitamin K.sub.1, mcg                                                                          100-140     11.90-17.24                                       Vitamin C, mg   350-450     41.67-55.42                                       Thiamin (B.sub.1), mg                                                                         2.66-4.6    0.32-0.57                                         Riboflavin (B.sub.2), mg                                                                      5.03-9.0    0.60-1.11                                         Pyridoxine (B.sub.6), mg                                                                      2.6-3.4     0.31-0.42                                         Vitamin B.sub.12, mcg                                                                         4.47-9.5    0.53-1.17                                         Pantothenic Acid, mg                                                                          15.4-24.0   1.83-2.96                                         Folic Acid, mcg 340-450     40.48-55.42                                       Niacin, mg      40.6-65     4.83-8.00                                         Biotin, mcg     350-460     41.67-56.65                                       Choline, mg      81-243      9.64-29.93                                       m-Inositol, mg  44.7-61     5.32-7.51                                         L-Carnitine, mg 35-60       4.17-7.39                                         Taurine, mg     60-80       7.14-9.85                                         Energy (kcal)   812-840                                                       ______________________________________                                         *assumes 24 kcal per fluid oz.                                           

The following Table III sets forth the lipid content (the only variable)used in the Control and Experimental products. It can be seen that thetwo formulas have the same total lipid amount, but differ principally inthe exchange of egg phospholipid for a portion of the medium chaintriglycerides. This substitution brings significantly higher levels ofphospholipids and choline as well as additional long chain PUFAs.

                  TABLE III                                                       ______________________________________                                        Lipids for Control and Experimental Formulas                                  Ingredient      Control     Experimental                                      ______________________________________                                        Lipid Blend     wt %        wt %                                              MCT*            50          41                                                coconut oil**   30          30                                                soy oil         20          20                                                egg phospholipid                                                                              0           9 (4.0 gm/L)                                      as choline                  75% of 9%                                         as ethanolamine             20% of 9%                                         soy lecithin    0.45 gm/L   0.0 gm/L                                          Cholesterol     ND.sup.+    ND.sup.+                                          Total Lipid (g/L)                                                                             44.1        44.1                                              ______________________________________                                         *MCT = medium chain triglycerides                                             **fractionated                                                                .sup.+ ND = none detected                                                

The Table IV sets forth the composite fatty acid profile for the Controland Experimental formulas. This represents the sum of the fatty acidcomponents of the egg lecithin and the Similac Special Care® formula.

                  TABLE IV                                                        ______________________________________                                        Average Fatty Acid Profiles in Weight %                                       Fatty Acid Control        Experimental                                        point of unsaturation                                                                    ω-3                                                                            other   ω-6                                                                           ω-3                                                                          other ω-6                          ______________________________________                                         6:0 - caproic    0.71               0.27                                      8:0 - caprylic   30.56              23.11                                    10:0 - capric     19.61              16.44                                    12:0 - lauric     9.69               10.24                                    14:0 - myristic   3.85               4.08                                     15:0 and 14:1     0.04               0.01                                     16:0 - palmitoleic                                                                              5.51               7.65                                     16:1 - palmitoleic                                                                              0.03               0.12                                     16:2              --                 --                                       17:0 - margaric   0.04               0.09                                     16:3              --                 --                                       16:4              --                 --                                       18:0 - stearic    2.68               3.89                                     18:1 ω-9 - oleic                                                                          8.31               11.25                                    18:2 ω-6 - linoleic 16.36            18.87                              18:3 ω-6 - linolenic                                                                              2.3              --                                 18:3 ω-3                                                                           2.24                 2.45                                          18:4 ω-6            --               0.02                               20:0 - arachidic  0.12               0.14                                     20:1 ω-9    0.04               0.09                                     20:2 ω-9    0.02               0.02                                     20:3 ω-9    --                 0.05                                     20:4 ω-6 - AA       --               0.41                               20:4 ω-3                                                                           --                   --                                            20:5 ω-3                                                                           --                   --                                            22:0 - behenic    0.07               0.12                                     22:5 ω-6            --               0.07                               22:5 ω-3                                                                           --                   0.07                                          22:6 ω-3 - DHA                                                                     --                   0.14                                          24:0              0.04               0.07                                     Total LC PUFA ω-3                                                                  0                    0.21                                          Total LC PUFA ω-6   0                0.48                               Total ω-3                                                                          2.24                 2.66                                          Total ω-6           18.66            19.37                              ______________________________________                                    

The inclusion of the egg phosphatide resulted in 0.21 weight percent ofthe total lipid blend as long chain ω-3 fatty acids and 0.48 weightpercent of the total lipid blend as long chain ω-6 fatty acids. Morespecifically, 0.14 weight percent of the total fat blend was DHA and0.41 weight percent of the total lipid blend was AA. Based onadministration of 100 kcal/kg/day for a 1 kg infant, this formulaprovides about 22 mg of AA and about 7 mg of DHA per day. Of course thisis just one possible embodiment of ratios within the invention.

EXAMPLE III

In this experiment, process variables were evaluated in an effort toreduce the organoleptic drawbacks associated with the use of eggphospholipids. The isolation of egg phospholipids useful in the presentinvention often results in an egg phosphatide that has somewhatobjectionable organoleptic properties for use in an infant formula.These can be improved yet further to provide a product that is notobjectionable to either the infant or the care giver. This process toimprove the final product is described below.

A number of nutritional formulas similar to Example II were preparedexcept that 6% by wt. of the fat blend was egg phospholipid which waspre-treated using various procedures. Egg phospholipid was dispersed ina portion of the oil blend described in Example II or in a portion ofthe water. The oil dispersions were unacceptable and could not be usedeven after heating to about 95° C. The dispersion of the phospholipidinto water from ambient to warm temperature was accomplished easily andis the preferred means of forming the water dispersion.

A master batch of 3% by wt. egg phospholipid dispersion was prepared byblending the phospholipid in 90° C. water for about 1 hour. A portion ofthis dispersion was passed through: (1) a de-aerator alone; (2) a carbonfiltration unit alone; (3) a de-aerator and a carbon filtration unitcombined; or (4) no treatment.

The activated carbon filtration unit contained 80 gms of activatedcarbon and the de-aeration unit was operated at a moderate vacuum (15in. Hg). The batch portions were passed through the filtration unit 3times and through the de-aerator once. When both techniques were used,the portion was passed through the filter first, then the de-aerator.The treated portions were then added to respective nutritional formulasjust prior to homogenization and sample packaging.

The samples were then initially evaluated for "flavor notes"(organoleptic properties) by a panel of trained evaluators. The resultsof the panel are set forth in Table V.

                  TABLE V                                                         ______________________________________                                        Infant Formula with Egg Yolk Phospholipids                                    Organoleptic Quality Results                                                                Flavor Notes *                                                  Treatment of Dispersion                                                                       Initial AA      AAL at 3 months                               ______________________________________                                        De-aeration alone                                                                             AA       1-2.5  AAL   2.5                                     Carbon Filtration alone                                                                       AA     1.5-2    AAL   2.5                                     De-aeration/Carbon Filtration                                                                 AA     2        AAL   2.5                                     No Treatment    AA     2.5-3    AAL   3                                       ______________________________________                                         * Flavor Notes                                                                AA = Arachidonic Acid                                                         AAL = Arachidonic Acid Lingering                                              + Scale                                                                       0.5 Very slight;                                                              1 Slight;                                                                     1.5 Slight to Moderate;                                                       2 Moderate;                                                                   2.5 Moderate to Strong; and                                                   3 Strong                                                                 

Surprisingly, the least aromatic sample was that which contained thedispersion that was passed through the de-aerator only. The dispersionthat was passed through the de-aerator and the carbon filter had thepoorest rating except for the control (no treatment of phospholipiddispersion).

EXAMPLE IV

Formulas prepared in accordance with Examples II and III were fed toinfants in a study conducted in the Neonatal Nursery of the Universityof Tennessee Newborn Center under the direction of Dr. Susan E. Carlsonwith financial support from Ross Products Division of AbbottLaboratories (Study AE78), NICHD grant RO1-HD31329, and National EyeInstitute grant RO1-EY08770. Research parameters included growth, neuraldevelopment, and visual acuity. Long chain PUFAs are believed to bephysiologically important for the development of the brain and eye, andare rapidly accumulated in fetal tissues in the last trimester ofpregnancy. Thus, pre-term infants do not accrete normal levels of longchain PUFAs relative to term infants.

Inclusion criteria: Entry into this clinical study was based on a "low"birth weight of less than 1500 gm (range 750-1375 gm) with no evidenceof cardiac, respiratory, gastrointestinal or other systemic disease. Theinfant also had no history of birth asphyxia or clinical complicationsof blood group incompatibility. The mothers of the enrolled infants hadno medical history of prenatal infections with proven adverse effects onthe fetus. Maternal substance abuse was an exclusion criteria. Allinfants initiated oral feedings by day 7 of life.

During the clinical study, a total of 120 infants were enrolled withinthe first 7 days of life. With the exception of one infant who wastransferred to another hospital shortly after enrollment (Control), allother infants (n-119) were cared for in the same hospital. Infants wereenrolled (randomized, blind) into 1 of 3 groups, two of which receivedthe Control formula during their hospitalization and one of whichreceived the Experimental formula (see Example II). Infants lost duringhospitalization were replaced by another infant assigned to the sametreatment group. Because of the study design, more infants were fed theControl formula. The total number fed the Control formula was more thantwice the number fed the Experimental formula.

Findings: An unanticipated finding was that a higher incidence ofnecrotizing enterocolitis (NEC) was seen in the Control Groups than theExperimental Group. Table VI groups the total number of neonatesaccording to treatment (Control v. Experimental) and sets forth thenumber of neonates in each group that developed NEC. NEC was consideredpresent or suspect when clinical signs and symptoms consistent with thisdisease, such as abdominal distention, gastric residuals, biliousvomiting, heme positive stools, presence of mucosa in stools, andpresence of C-reactive protein at ≧0.5 mg/dL (Pourcyrous et al.,"Significance of Serial C-reactive Protein Responses in NeonatalInfection and Other Diseases", Pediatr., 1993, 92:431-435). NEC wasconfirmed in 15 of the Control infants and only 1 of the Experimentalgroup.

                  TABLE VI                                                        ______________________________________                                        Results of Clinical Study                                                                  Control                                                                             Experimental                                               ______________________________________                                        NEC*           15       1                                                     no NEC         70      33                                                     TOTAL          85      34                                                     ______________________________________                                         *or suspected of NEC                                                     

Statistical analysis of this data, using Fisher's exact test (twotailed), shows that the number of infants confirmed with NEC in theControl treatment group(s) was significantly greater (p=0.039) than thenumber of infants in the Experimental treatment group having NEC.

EXAMPLE V

In this experiment, the inclusion of AA and DHA into parenteral(intravenous feedings) administration of nutrition, is evaluated. Theparenteral solution can contain the various components known in the artwith the AA and DHA being supplied in the form of a phospholipid,triglycerides or the methyl esters. The AA and DHA may be the soleactive ingredients admixed with conventional parenteral vehicles andexcipients or, more preferably, the AA and DHA is included a parenteralformula intended to supplement or supply the total nutritional supportof the infant. Typical parenteral nutritional solutions contain levelsof lipids resulting in about 2 g/kg/day. The level of AA and DHA in thelipid blend should preferably result in the administration of from 10 to30 mg/kg/d of AA and 3 to 15 mg/kg/d for DHA.

EXAMPLE VI

In this experiment, the egg lecithin of the experimental formula ofExample II is replaced by soy lecithin at the approximately ten-foldhigher levels than found in the control formula. Soy lecithin, likeother phospholipids derived from vegetable sources, contain nolong-chain polyunsaturated acids; however, the polar nature ofphospholipids and their ability to be readily incorporated into theintestinal mucosa may afford a protective effect on the intestinallining, thereby producing results comparable to those of theexperimental formula of Example II. Additionally, soy lecithin containslinoleic acid (18:2ω-6 --a dietary essential fatty acid precursor to AA)and linolenic acid (18:3ω-3--a dietary essential fatty acid precursorDHA).

EXAMPLE VII

In this experiment, the use of phospholipids containing AA and DHA ininfant formula is compared to triglycerides containing AA and DHA. Theformula of Example II is compared to a similar infant formula whereinthe egg phospholipid is replaced with a mixture of single cell microbialtriglycerides containing comparable levels of AA and DHA.

Healthy full term infants are enrolled in a clinical evaluation tomeasure the blood serum levels of AA and DHA following enteraladministration. It is expected that infants fed the phospholipid formulawill achieve blood serum levels of AA and DHA more closely resemblingthose of breast fed infants than the control formula containing AA andDHA in triglyceride form. This experiment should demonstrate thatphospholipids containing AA and DHA are a preferred form ofadministration over triglycerides containing AA and DHA. Thus, improvedenteral formulas and methods for increasing AA and DHA blood serumlevels are contemplated herein.

Modifications and alternative embodiments of the compositions andmethods of the present invention will be apparent to those skilled inthe art in view of the foregoing description. Accordingly, thisdescription is to be construed as illustrative only and is for thepurpose of teaching those of skill in the art the manner of carrying itout. The full scope of the invention for which protection is sought isdefined in the appended claims.

What is claimed is:
 1. A method for reducing the incidence of necrotizing enterocolitis in an infant, said method comprising administering to an infant which is susceptible to necrotizing enterocolitis an effective amount of a nutritional composition containing protein, carbohydrate and phospholipids to provide at least 1.0 mg of ω-6 polyunsaturated fatty acids per kg per day.
 2. The method according to claim 1 wherein said at least one ω-6 polyunsaturated fatty acid includes arachidonic acid.
 3. The method according to claim 2 wherein said administration is performed enterally.
 4. The method according to claim 1 wherein said at least one ω-6 polyunsaturated fatty acid is administered in combination with at least one ω-3 polyunsaturated fatty acid.
 5. The method according to claim 4 wherein said ω-3 polyunsaturated fatty acid comprises docosahexaenoic acid.
 6. The method according to claim 4 wherein said ω-6 polyunsaturated fatty acid comprises arachidonic acid and said ω-3 polyunsaturated fatty acid comprises docosahexaenoic acid.
 7. The method according to claim 6 wherein said administration is performed by feeding a sufficient amount of an enteral composition containing arachidonic acid and docosahexaenoic acid to deliver to said infant about 1.0 to about 60 mg per kg per day of arachidonic acid and about 0.25 to about 35 mg per kg per day of docosahexaenoic acid.
 8. The method according to claim 7 wherein the weight ratio of arachidonic acid to docosahexaenoic acid ranges from about 2 to about
 4. 9. The method according to claim 7 wherein the enteral composition additionally comprises vitamins and minerals.
 10. The method according to claim 4 wherein said ω-6 polyunsaturated fatty acid and said ω-3 polyunsaturated fatty acid are long chain fatty acids independently selected from one or more sources selected from the group consisting of egg lecithin, fungal oils, algal oils and marine oils.
 11. The method according to claim 4 wherein said ω-6 polyunsaturated fatty acid and said ω-3 polyunsaturated fatty acid are derived from genetically modified oil-bearing plants.
 12. The method according to claim 4 wherein said ω-6 polyunsaturated fatty acid and said ω-3 polyunsaturated fatty acid are in the form of phospholipids.
 13. The method according to claim 1 comprising feeding to an infant which is susceptible to necrotizing enterocolitis a sufficient amount of an enteral formula containing: protein, carbohydrate and phospholipids, said phospholipids providing arachidonic acid and docosahexaenoic acid in concentrations to deliver to said infant from about 5 to about 40 mg per kg per day of arachidonic acid and from about 1.5 to about 20 mg per kg per day of docosahexaenoic acid, wherein the weight ratio of arachidonic acid to docosahexaenoic acid ranges from about 2 to about
 4. 14. A method for decreasing the incidence of necrotizing enterocolitis in an infant, said method comprising feeding to said infant a sufficient quantity of an enteral nutritional composition containing protein, carbohydrate and phospholipids to provide at least 2.0 mg of ω-6 polyunsaturated fatty acids per kg per day.
 15. The method according to claim 14 wherein said feeding further provides at least 0.5 mg ω-3 polyunsaturated fatty acids per kg per day.
 16. The method according to claim 15 wherein said feeding provides at least 2.0 mg arachidonic acid and at least 0.5 mg docosahexaenoic acid per kg per day.
 17. The method according to claim 16 wherein said feeding provides from about 5 to about 40 mg arachidonic acid per kg day.
 18. The method according to claim 17 wherein said enteral composition comprises egg phospholipid.
 19. The method according to claim 15 wherein said enteral composition comprises egg phospholipid.
 20. The method according to claim 19 wherein said enteral composition further comprises vitamins and minerals.
 21. A method for decreasing the incidence of necrotizing enterocolitis in an infant, said method comprising administering to an infant susceptible to necrotizing enterocolitis a sufficient quantity of a nutritional composition containing protein, carbohydrates and phospholipids to provide between about 60 and about 2400 μmoles of phospholipids per kg day.
 22. The method of claim 21 comprising administering a sufficient quantity of said nutritional composition to provide between about 200 and about 1500 μmoles of phospholipids per kg per day.
 23. The method of claim 21 wherein said phospholipids are derived from egg lecithin.
 24. The method of claim 21 wherein said phospholipids are administered in combination with one or more polyunsaturated fatty acids selected from the group consisting of arachidonic acid and docosahexaenoic acid.
 25. The method of claim 24 wherein said phospholipids are administered in combination with arachidonic acid and docosahexaenoic acid to give from about 200 to about 1500 μmoles of phospholipid; from about 5.0 mg to about 40 mg arachidonic acid; and from about 1.5 mg to about 20 mg docosahexaenoic acid per kg per day.
 26. The method of claim 21 wherein said phospholipids are administered enterally.
 27. A method for decreasing the incidence of necrotizing enterocolitis in an infant, said method comprising administering to an infant susceptible to necrotizing enterocolitis a sufficient quantity of a nutritional composition containing protein, carbohydrates and phospholipids to provide between about 60 and about 1800 μmoles of choline per kg per day.
 28. The method of claim 31 comprising administering a sufficient quantity of said nutritional composition to provide between about 150 and about 1200 μmoles of choline per kg per day.
 29. The method of claim 27 wherein said choline is in the form of phosphatidylcholine.
 30. The method of claim 29 wherein said phosphatidylcholine is derived from egg lecithin.
 31. The method of claim 27 wherein said choline is administered in combination with one or more polyunsaturated fatty acids selected from the group consisting of arachidonic acid and docosahexaenoic acid.
 32. The method of claim 31 wherein said choline is administered in combination with arachidonic acid and docosahexaenoic acid to give from about 150 to about 1200 μmoles of choline; from about 5.0 mg to about 40 mg arachidonic acid; and from about 1.5 mg to about 20 mg docosahexaenoic acid per kg per day.
 33. The method of claim 27 wherein said choline is adminstered enterally. 